Acute diarrheal disease due to transient colonization of the small bowel by enterotoxigenic strains of Escherichia coli (E. coli or ETEC) is a major health problem of global scope for both humans and for animal husbandry. These organisms, together with rotavirus, are the principal cause of the often fatal acute diarrhea that is common among infants living in underdeveloped countries and among neonatal animals, particularly lambs and piglets. ETEC strains are also the usual cause of acute diarrhea among persons from temperate zones who travel to the tropics, and may be responsible for sporadic or epidemic episodes of diarrhea among children and adults living in either temperate or tropical areas.
The disease caused by ETEC is mediated by the release of two enterotoxins, either singly or together. The large molecular weight, antigenic heat-labile toxin (LT) has been purified to homogeneity and its subunit structure characterized as five B subunits which attach the holotoxin to the specific GM.sub.1 ganglioside receptors on the mucosal surface, and a single A subunit which stimulates intracellular adenylate cyclase activity, thus evoking fluid and electrolyte secretion.
The low molecular weight, heat-stable toxin (ST) produced by ETEC strains of human or porcine origin has also recently been purified. Preparations of ST have a relatively high content of half-cystine, cause secretion by stimulating guanylate cyclase and are haptenic as evidenced by their capacity to raise an antitoxin response in animals immunized with the toxin coupled to a large molecular weight carrier.
The most practical approach for the prevention of ETEC-induced diarrhea would be an immunization program that provides protection against heterlogous ETEC serotypes that produce either or both of the LT or ST enterotoxins. Immunization with either the biologic LT or the biologic ST toxin evokes an antitoxin response in experimental animals that protects against homologous and heterlogous serotypes of strains that produce the specific toxin used for immunization. Immunization with the LT whole toxin or its B subunit yields protection against viable heterlogous strains that produce this toxin alone (LT.sup.+ /LT.sup.-) or together with ST (LT.sup.+ /ST.sup.+), but not against those which make just ST (LT.sup.- /ST.sup.+).
Immunization with biologic ST coupled to a large molecular weight carrier arouses serum antibodies that passively neutralize the secretory effect in the suckling mouse model of ST produced by heterlogous strains. Immunization also provides protection against direct challenge with viable heterlogous LT.sup.- /ST.sup.+, but not LT-producing strains. Neither of these toxins is suitable for immunization when given along, however, in view of their toxicity, their failure to provide protection against strains which produce the other toxin form, and the fact that the large molecular weight carriers that have been used to render the haptenic biologic ST molecule antigenic are unsuitable for human use.
Klipstein et al., Infect. Immun., 37: 550-557 (1982) have reported the development of a vaccine made by conjugating the biologic ST toxin to the LT toxin by means of the carbodiimide reaction. As a result of that reaction, biologic ST acquires antigenicity when coupled to the large molecular weight LT carrier, while both cross-linked toxins retain most of ther antigenicity but loose most of their toxix properties. Rats immunized with the vaccine so produced were strongly protected against challenge with either LT or biologic ST and with viable ETEC strains which produce those toxins.
A semi-pure preparation of biologic ST was used for that vaccine because of the relatively low yield of pure biologic ST obtained by the available purification techniques which involve multiple chromatographic separation steps. The inclusion of the heterogeneous material in the vaccine may preclude its use for human immunization, however.
The present invention, relating to the synthetically produced ST, has overcome the problem of using ST derived from natural sources in that synthetic ST can be made in large quantities and in purified form, and has properties similar to those described for pure ST obtained by bacterial growth of a human ETEC strain. [Staples et al., J. Biol. Chem., 255: 4716-4721 (1980); and Chan et al., J. Biol. Chem., 256: 7744-7746 (1981)].
At least two types of ST have been identified by their physical properties. The first type known as ST I (also referred to as STa) is soluble in methanol and is active in the suckling mouse model. The second type, ST II (also referred to as STb) is methanol insoluble and not active in the suckling mouse model, but is active in ligated pig ileal loops.
Among the ST I polypeptides, at least three similar polypeptides, or determinant domains of those polypeptides, have been identified, and their amino acid sequences determined. These three types of ST I are referred herein as (i) ST Ia which was initially found in a bovine E. Coli strain and a portion of which is also encoded in porcine strains, (ii) that designated ST Ib from a human isolate of E. coli and (iii) ST Ic also isolated from human-infecting E. coli.
The nucleotide sequence coding for the ST Ia polypeptide has been determined. Translation of the nucleotide sequence into a polypeptide amino acid sequence leads to a polypeptide that contains 72 amino acids capped at the carboxy-terminus with a tyrosine group [So et al., Proc. Natl. Acad. Sci. USA, 77: 4011-4015 (1980)]. The ST Ic polypeptide is though to also contain 72 amino acids as well as several homologous domains with the ST Ia polypeptide. The ST Ib polypeptide is reported to contain only 18 amino acids ([Chan et al., J. Biol. Chem., 256: 7744-7746 (1981)].
The 18 amino acids of the ST Ib polypeptide (18-mer) show great homology to amino acids 55 through 72 for the polypeptide of ST Ia. The homologous, almost identical, region is illustrated hereinbelow, beginning at amino acid number 55, from left to right and in the direction of amino-terminus to carboxy-terminus, of the ST Ia polypeptide:
ST Ia: AsnThrPheTyrCysCysGluLeuCysCys PA1 ST Ib: AsnThrPheTyrCysCysGluLeuCysCys PA1 ST.sub.h AsnSerSerAsnTyrCysCysGluLeuCysCys PA1 ST.sub.p - - - AsnThrPheTyrCysCysGluLeuCysCys PA1 "e" is zero when "a" is zero, PA1 "d" is zero when "b" is zero, and PA1 "f" is zero when "c" is zero; PA1 each of "g" and "k" is zero when "a" is zero, PA1 each of "h" and "j" is zero when "b" is zero, and PA1 each of "i" and "l" is zero when "c" is zero; and
AsnProAlaCysAlaGlyCysTyr
TyrProAlaCysAlaGlyCysAsn
More recent reports by Takeda et al., Abstracts, 19th Joint Conference US-Japan Cooperative Medical Science Program, Cholera Panel, 87-88 (1983) and Ikemura et al., Chem. Letters, (Chem. Soc. Japan), 101-104 (1983) have indicated the presence of further polypeptide sequences for this 18-mer polypeptide. Those workers referred to the ST molecule obtained from human and porcine strains of ETEC as ST.sub.h and ST.sub.p, respectively. The amino acid residue sequences reported by those workers, from left to right and in the direction from amino-terminus to carboxy-terminus, are:
AsnProAlaCysThrGlyCysTyr
AsnProAlaCysAlaGlyCysTyr
As can be seen from a comparison of both of the above sets of sequences; i.e., ST Ia, ST Ib, ST.sub.h and ST.sub.p, a great deal of homology is shared among the carboxy-terminal fourteen residues of each of the four sequences shown.
Those workers also reported a synthesis of ST.sub.h. Solution methods of synthesis were used to prepare the blocked polypeptide. Blocking groups were removed with hydrogen fluoride, and the Cys mercapto groups (thiols) were air oxidized. Air oxidation was carried out at a polypeptide concentration of 10.sup.-5 molar in distilled water adjusted to a pH value of 8.0 with aqueous ammonia. Oxidation was continued until free thiol groups disappeared.
Biologic activity of the synthetic ST.sub.h in a suckling mouse assay was reported to be the same as that for native toxin. Toxicity of the synthetic material was reported to be neutralized by antisera against the native toxin.
Examination of the above four 18-amino acid polypeptide sequences also reveals that six half-cystine (Cys) residues that are present. Oxidation of those half-cystine residues to cystine residues containing intramolecular disulfide bonds in the naturally occuring enterotoxin is thought to lend the observed heat stability to that material.
It is further noted, however, that while cystine disulfide bonds are known to be present in biologic ST, it is not known which pairs of half-cystine residues combine to form the three disulfide bonds that are present in the native ST molecule. Those three sulfide bonds can theoretically be formed from fifteen different combinations of the six Cys residues present.
Staples et al., supra, have shown that the disulfide linkages of biologic ST are required for biological activity of the toxin. Thus, chemical reduction to form half-cystines or performic acid oxidation to cysteic acid was shown to destroy the biological activity of the toxin. In addition, Chan et al., supra, have reported that the first four residues from the amio-terminus of the homologous 18-amino acids of the above sequence of St Ib are not required for biological activity. Thus, biological activity was obtained from the amino acid-containing polypeptide comprising the above carboxy-terminal 14 amino acids and their disulfide bonds.
Aimoto et al, Biochem. Biophys. Res. Chem., 112: 320-326 (Apr. 15, 1983) have reported on the synthesis of the carboxy-terminal fourteen amino acid residues of the beforedescribed ST.sub.h. That synthetic molecule was reported to have biologic activity 2-5 times that of the native ST.sub.h on a molar basis, using a suckling mouse assay.
In an oral presentation on Aug. 29, 1982 by Duflot et al., Proceedings European Peptide Symposium: 683-686, published in Berlin in June of 1983, those workers reported the synthesis of a porcine and human ST 18-mer polypeptides having their Cys mercapto groups blocked (S-blocked) with acetamidomethyl groups. Those amino acid residue sequences were purportedly identical to the sequences reported by So et al, supra, for ST Ia and by Chan et al., supra, for ST Ib. However, the seventh amino acid residue from the amino-terminus of the sequences reported by Duflot et al. was a glycine residue (Gly), while that residue in the beforedescribed sequences is a glutamic acid residue (Glu).
Duflot et al. reported that immunization of micr or rabbits with their S-blocked porcine ST toxin coupled to tetanus toxoid or ovalbumin produced antibodies that recognized the natural or the synthetic toxins equally. Substantially no biologic activity in the suckling mouse assay was reported for the S-blocked, porcine, synthetic polypeptide toxin. Those authors reported the lack of biologic activity to be due to the absence of intramolecular disulfide bonds in the S-blocked molecule, which is in keeping with the prior report of Staples et al., supra.